Health of the ocular surface is dependent on tear fluid secretions from the lacrimal gland. The lacrimal acinar cells comprising the lacrimal gland are polarized and highly differentiated tear secreting cells that adhere to a complex periacinar basement membrane. The bulk of the apical cell cytoplasm contains large secretory granules packed with tear proteins. Known tear proteins include: lysozyme, which plays a prominent bactericidal role on the corneal surface; lactoferrin, which functions as both a bactericidal agent and as a potential inhibitor of complement activation; secretory component, which regulates the transcellular movement of IgA into acini lumen where it acts on the corneal surface to inhibit bacterial adhesion; and tear lipocalins (tear-specific prealbumin) and growth factors TGFα, TGFβ and EGF the functions of which are not known. In rats, peroxidase is a tear component which has served as a convenient marker in experimental studies. Tears not only have an important bactericidal role, they also keep the cornea clean and lubricated and are important for the well-being of the corneal epithelium.
The surface of the eye is one of the most accessible and vulnerable tissues. Corneal epithelial cells confront environmental insults constantly including: UV irradiation, widely varying air temperature fluxes, pollutants, bacteria and other microbial organisms. The tear fluid which bathes the corneal surface is the most likely source of cytoprotective and anti-inflammatory agents since the cornea lacks blood supply, unlike other tissues where blood vessels supply such agents. Indeed, tear fluid is rich in bactericidal proteins. Dry Eye patients suffering insufficient tear production are subject to corneal ulceration, infection or inflammation. Similar symptoms can be generated by extended contact lens use, since volume of tear supply is limited.
When lacrimal acinar cell tear output is collectively deficient, ‘Dry Eye’ (also known as keratoconjunctivitis sicca [KCS]), is the result. Dry Eye is a common ocular manifestation of Sjogren's syndrome, an autoimmune disease with unknown etiology that affects millions of people worldwide. Most commonly affected are post-menopausal women with varying degrees of severity. If untreated, Dry Eye can lead to corneal abrasion, ulceration, bacterial infection and loss of vision.
Molecular mechanisms underlying the pathogenic decline of secretory output by the main lacrimal gland are potentially multiple. Lacrimal glands of Sjogren's syndrome patients contain foci of B and T lymphocytes whose pathogenic expansion, possibly due to viral insult, can destroy lacrimal acini. However, acinar volume loss often appears insufficient relative to the theoretical overcapacity of the main lacrimal gland. Estimates suggest a potential secretory output up to ten-fold greater than is required to maintain a normal aqueous tear film layer. Other mechanisms therefore warrant attention, such as aberrant secretion of one or several common cytokines that may directly or indirectly alter lacrimal acinar cell function and/or lead to a decline in neural innervation. Novel autocrine/paracrine factor(s) released by lacrimal acinar cells into the tear film may be required for the health of the lacrimal secretory machinery, ductal system and corneal epithelium. The periacinar basement membrane is also required for normal secretory function, in part via ‘BM180’ whose apparent synergy with laminin-1 promotes stimulated tear secretion. Alteration of each of these factors, together or independent of hormonal changes, could contribute to decreased secretory capacity.
The lacrimal-corneal axis is a fundamental regulator of ocular health and plays a key role in ocular surface inflammation associated with Dry Eye Syndromes and corneal injury. A host of mediators are implicated in the development and progression of corneal inflammation, such as the proinflammatory cytokines TNF-α, IL-1β, IL-6 and the chemokine IL-8. Also involved are the arachidonic acid-derived eicosanoids which are produced by the activity of cyclooxygenases (primarily PGE2), lipooxygenases (12 (s)-HETE) and cytochrome P450 (12 (r)-HETE). Recent studies on lacritin mechanisms of action indicate converging PKCα and NFkB signaling pathways suggesting that lacritin may have a key anti-inflammatory role on the ocular surface. Recent clinical studies support this hypothesis. Comparison of tear proteins from 19 patients suffering from Blepharitis (inflammation of the lid) vs 27 healthy volunteers revealed lacritin to be decreased by 56% in patients.
Regulation of Slowly Renewing Epithelia
Epithelial renewal underpins organogenesis, homeostasis, and tumorigenesis in a growth and adhesion factor-modulated manner. Proliferation of slowly cycling populations of stationary, often sequestered, stem cells and their migratory ‘transit amplifying’ cellular offspring is dependent on compartmentalized extracellular cues that include β1 integrin adhesivity (Alonso, L., et al., Genes Dev., 2003, 17:1189-200) to basement membrane laminin (Li, S., et al., Dev. Cell, 2003, 4:613-624), and involvement of the Wnt family of mitogens, which display a particular proclivity towards renewing cell compartments (Pinto, D., et al., Genes Dev., 2003, 17:1709-1713; Kuhnert, F., et al., Proc. Natl. Acad. Sci. USA, 2004, 101:266-271).
In small intestine, Wnts secreted by crypt mesenchyme and epithelia act in a paracrine or autocrine manner to generate a proliferative zone seeded by a single stem cell in each crypt. Crypts are spatially separated but contiguous with outward protruding villi in which cessation of Wnt secretion terminates cell cycle progression and promotes hedgehog-dependent differentiation of the absorptive epithelial, enteroendocrine or goblet cells necessary for intestinal function. In skin, stem cells reside in the hair follicle bulge where continuous FGF-18 and BMP-6 proliferative suppression is thought to balance with positive proliferative pressure by Wnt signaling until disruptive skin injury requires replenishment of proliferative cells at the base of the epidermis. The equivalent unit of epithelial renewal in skin comprises one hair follicle and adjacent epidermis. Similar principles may be in play on the surface of the eye since Wnt signaling promotes corneal epithelial proliferation in response to injury. Corneal stem cells encircle the periphery of the cornea to contribute progeny, which migrate centrally as the proliferative basal layer of the stratified corneal epithelium. The stem cell niche is thus a key characteristic of rapidly renewing epithelia.
How turnover is regulated in non-rapidly renewing or persistent epithelia is more challenging to address, because a discrete stem cell niche appears to be absent. Epithelia displaying slow turnover under normal conditions comprise most adult organs of the body, including exocrine and endocrine glands, kidney, and liver. The recently discovered human protein ‘lacritin’ may offer new insights. Lacritin is secreted by acinar cells in adult lacrimal gland and a subpopulation of ductal cells in salivary glands; and has been detected by non-histological methods in thyroid and mammary glands, but not elsewhere.
Lacrimal, salivary, and mammary glands are exocrine glands in which long ductal ingrowths carry protein products from secretory acini to the outside. In salivary glands, the highly proliferative intercalated ductal cell acts like a stem cell or transit amplifying cell to populate upstream acinar and downstream ductal cell types. Importantly, all epithelial cell types also appear to be slowly cycling (Man, Y. G., et al., Anat. Rec., 2001, 263:202-214), much like adult mammary gland in which stem cells are located throughout the epithelium in different early stages of differentiation. The lacrimal gland has not been similarly examined, but slow proliferative capacity throughout the epithelium appears to be a common property of exocrine glands and other non-rapid renewing epithelia.
Many epithelial cells contact lacritin as it flows downstream from acini or ducts, a simple distribution mechanism whose intermittent quality could play a large role in regulating the slow renewal of this exocrine glandular epithelium. Also potentially influenced are the rapidly renewing epithelial cells on the surface of the eye and mouth where lacritin is deposited. Supporting this hypothesis are in vitro studies showing recombinant lacritin to be capable of promoting lacrimal acinar cell secretion, human salivary ductal cell proliferation, and calcium mobilization by human corneal epithelial cells (Sanghi, S., et al., J. Mol. Biol., 2001, 310:127-139). Thus in the absence of a particular stem cell niche, release of lacritin defines an intermittent proliferative field that spreads through the non-rapidly renewing epithelia of lacrimal and salivary glands. Since other non-rapidly renewing epithelia have luminal structures, proliferative fields may be a general principle of non-rapidly renewing or persistent epithelia.
There is a long felt need in the art for a method to promote survival of ocular epithelial cells, to prevent and treat ocular inflammation, and to enhance corneal repair following injury or surgical treatment. The present invention satisfies these needs.